Multiphase Bioreactor Design

After much initial interest in two-liquid phase biocatalysis it has become clear that there
are three primary applications for this technology. These are for biotransformations
involving:

● Poorly water-soluble reactants which are inhibitory or toxic to the biocatalyst.
● A difference in the water-solubility of reactant(s) and/or product(s).
● Poorly water-soluble reactants which are solids and inhibitory or toxic and therefore
cannot be fed easily to the reactor.

Alternative reactor technologies do now exist in a number of cases. For example solid,
poorly water-soluble reactants can be used in slurry reactors where the properties of the
biocatalyst allow this (Kasche et al., 1995; Michielsen et al., 2000). Likewise in order to
overcome downstream liquid-liquid emulsion separation problems the use of a solid
hydrophobic adsorbent can be used as a reservoir for the reactant (Vicenzi et al., 1997).
Although limited by the capacity of the resin this is a very effective technique providing
the biocatalyst can tolerate the reactor environment. In many examples however there
remain very clear process advantages to using two-liquid phase biocatalysis. In these
cases the productivity limitation frequently lies in the lack of tolerance of the biocatalyst
to the dissolved levels of organic phase or the presence of the liquid-liquid interface.
These are issues needing to be addressed in future research.

Designer Biocatalysts

Developments in molecular biology now enable a number of possibilities for biocatalysis
in two-liquid phase media. First, the identification of genes harbouring a particular
activity, together with cheap expression systems (for use at scale) mean that cloning the
enzymes from the natural host to another is now possible. The choice of host to date has
largely been based on the knowledge of genetics and ability to overexpress effectively in
the new host. It is clear that new opportunities are now possible to clone a desired
enzyme activity into a more solvent tolerant host. Other properties to be cloned for might
be a reduction in secreted or lysed material (implicated in mutiphasic media) or cell
softening which leads to particular problems for downstream processing (e.g. via
centrifugation). Directed evolution techniques (Arnold, 1996) will enable more solvent
tolerent enzymes to be developed but more crucially high activities with non-natural
substrates which will mean less catalyst is required. Difficulties in subsequent
downstream separation often show a close correlation with the amount of catalyst
present.

Designer Solvents

Recent work in our laboratories has established the use of room temperature ionic liquids
as direct replacements for organic solvents in two-phase biocatalytic processes (Cull et
al., 2000). Ionic liquids such as 1-butyl-3-methylimidazolium hexafluorophosphate,
[bmim][PF 6 ], are, as their name implies, solutions composed entirely of ions and have
been shown to be non-toxic to a whole cell Rhodococcus biocatalyst. Ionic liquids have,
among a unique set of chemical and physical properties (Seddon, 1997), effectively no
measurable vapour pressure, which makes them ideal replacements for volatile,

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